Lactic Acid

Lactic Acid Bacteria Biodiversity and

Edited by

Wilhelm H. Holzapfel School of Life Sciences, Handong Global University, Pohang, Gyeongbuk, South Korea; Insheimer Strasse 27, Rohrbach, Germany Brian J.B. Wood Formerly Reader in Applied Microbiology, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK This edition first published 2014 © 2014 by John Wiley & Sons, Ltd

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Library of Congress Cataloging-in-Publication Data : biodiversity and taxonomy / edited by Wilhelm Holzapfel. pages cm Includes bibliographical references and index. ISBN 978-1-4443-3383-1 (cloth) 1. Lactic acid bacteria. 2. Biodiversity. 3. Microbial diversity. 4. Lactic acid bacteria – Classification. 5. Lactic acid bacteria – Physiology. 6. Microbiological chemistry. 7. Lactic acid bacteria – Molecular aspects. I. Holzapfel, W. H. QR121.L3335 2014 579.3′5– dc23 2013028930

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12014 Contents

List of contributors xiii

Acknowledgements xv

List of abbreviations xvi

Abbreviations for genera and note on pronunciations xix

1 Introduction to the LAB 1 Wilhelm H. Holzapfel and Brian J.B. Wood 1.1 The scope 1 1.2 A little history 7 1.3 Where are the boundaries? 9

2 Physiology of the LAB 13 Akihito Endo and Leon M.T. Dicks 2.1 Metabolism 13 2.2 Energy transduction and solute transport 20

3 Phylogenetics and systematics 31 Peter Vandamme, Katrien De Bruyne and Bruno Pot 3.1 Introduction 31 3.2 Phylogeny and polyphasic taxonomy of LAB 34 3.3 Conclusions and perspectives 39

4 Overview of the ecology and biodiversity of the LAB 45 Giorgio Giraffa 4.1 Introduction 45 4.2 LAB ecology, diversity and metabolism 45 4.3 Importance of LAB in food and feed ecology and biotechnology 46 4.4 LAB as functional cultures 48 4.5 LAB with health-promoting properties 50 4.6 Concluding remarks 51

5 Comparative genomics of Lactobacillus and other LAB 55 Trudy M. Wassenaar and Oksana Lukjancenko 5.1 Introduction 55 5.2 Selection of LAB genomes for comparative analysis 57 vi CONTENTS

5.3 Numerical comparisons of the selected genomes 58 5.4 Phylogeny of the 16S rRNA gene extracted from the genomes 63 5.5 Pan-genome and core genome of protein genes 63 5.6 Comparison of gene function categories 66 5.7 Conclusions 68

Section I The family 71 Paul A. Lawson 6 The 75 Paul A. Lawson 6.1 Introduction and historical background 75 6.2 Description of the genus Abiotrophia 76 6.3 Differentiation of Abiotrophia from other genera 76 6.4 Isolation, cultivation, ecology and medical importance 76 6.5 Species descriptions 78

7 The genus 81 Paul A. Lawson 7.1 Introduction and historical background 81 7.2 Description of the genus Aerococccus 81 7.3 Differentiation of Aerococcus species from other genera 82 7.4 Differentiation of species of the genus Aerococcus from one another 83 7.5 Isolation, cultivation, ecology and medical importance 84 7.6 Species descriptions 86

8 The genus 91 Lesley Hoyles 8.1 Introduction 91 8.2 Differentiation of Facklamia species from other genera 91 8.3 Ecological, medical and industrial relevance of Facklamia species 92 8.4 Antimicrobial susceptibilities of members of the genus Facklamia 94 8.5 Differentiation between species of the genus Facklamia 95 8.6 Descriptions of the genus Facklamia and its species 95

9 Minor genera of the Aerococcaceae (, , , )99 Melanie Huch, Cho Gyu-Sung, Antonio Gálvez and Charles M.A.P. Franz 9.1 Historical background 99 9.2 Phenotypic differentiation of the minor genera of the Aerococcaceae from other genera 100 9.3 Genotypic delineation of the minor genera of the Aerococcaceae 101 9.4 Isolation, cultivation, ecology and medical importance 102 9.5 Description of the minor genera of the Aerococcaceae and list of species 102

Section II The family 107 Elena V. Pikuta 10 The genus Carnobacterium 109 Elena V. Pikuta and Richard B. Hoover 10.1 Historical background and chronology of nomenclature 109 10.2 Definition of the genus Carnobacterium 110 10.3 Relationship to other groups 111 10.4 Future perspectives for characterization 112 10.5 Techniques and growth requirements for cultivation 112 10.6 Biodiversity 112 10.7 Importance of the genus and particular species 113 10.8 Other applications and future perspectives 115 10.9 Description of species 115 CONTENTS vii

11 The genus Marinilactibacillus 125 Morio Ishikawa and Kazuhide Yamasato 11.1 Introduction 125 11.2 General and taxonomic characters 125 11.3 Phylogenetic affiliation of Marinilactibacillus species 126 11.4 Physiological properties 127 11.5 Differentiation of Marinilactibacillus from other related species 127 11.6 Lactic acid fermentation and aerobic metabolism of glucose 127 11.7 Ecology and isolation methods 129 11.8 Description of the species of the genus Marinilactibacillus 132

12 The genus Trichococcus 135 Elena V. Pikuta and Richard B. Hoover 12.1 Historical background and chronology of nomenclature for the Trichococcus species 135 12.2 Definition of the genus Trichococcus 136 12.3 Relationship to other genera within the Carnobacteriaceae and other LAB families 136 12.4 Future taxonomic perspectives 139 12.5 Techniques and growth requirements for cultivation of Trichococcus species 139 12.6 Biodiversity 139 12.7 Importance of the genus and particular species 140 12.8 Species descriptions 141

13 The genus Alkalibacterium 147 Isao Yumoto, Kikue Hirota and Kenji Nakajima 13.1 Introduction 147 13.2 Taxonomy 148 13.3 Description of the genus 148 13.4 Enrichment and isolation procedures 148 13.5 Natural habitats 149 13.6 Acid production 150 13.7 Identification of Alkalibacterium species 150 13.8 Overview of the current situation for this genus 150 13.9 Description of species 153 13.10 Concluding remarks 156

14 Minor genera of the Carnobacteriaceae: Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes, Bavariicoccus, Desemzia, Dolosigranulum, Granulicatella, Isobaculum and Lacticigenium 159 Ulrich Schillinger and Akihito Endo 14.1 Introduction 159 14.2 Taxonomy 159 14.3 Biodiversity of each genus 162 14.4 Practical importance 163 14.5 Species descriptions 164

Section III The family 171 Pavel Švec and Charles M.A.P. Franz 15 The genus Enterococcus 175 Pavel Švec and Charles M.A.P. Franz 15.1 Historical background and chronology of nomenclature 175 15.2 Phenotypic differentiation of the genus Enterococcus 178 15.3 Genotypic delineation of the genus Enterococcus 178 15.4 Phylogenetic structure within the genus Enterococcus 179 15.5 Isolation and cultivation 179 15.6 Identification of Enterococcus spp. 179 15.7 Importance of the genus and particular species 182 15.8 Species of the genus Enterococcus 186 viii CONTENTS

16 The genus Tetragenococcus 213 Annelies Justè, Bart Lievens, Hans Rediers and Kris A. Willems 16.1 Introduction 213 16.2 Phenotypic characteristics of the genus Tetragenococcus 215 16.3 Genotypic characteristics of the genus Tetragenococcus 217 16.4 Industrial relevance of the genus Tetragenococcus 221 16.5 Description of species 222

17 The genus Vagococcus 229 Paul A. Lawson 17.1 Introduction and historical background 229 17.2 Description of the genus Vagococcus 229 17.3 Differentiation of Vagococcus species from other genera 230 17.4 Differentiation of species of the genus Vagococcus from one another 231 17.5 Isolation, cultivation, ecology and medical importance 231 17.6 Species descriptions 232

18 Minor genera of the Enterococcaceae (Catellicoccus, Melissococcus and Pilibacter) 239 Leon M.T. Dicks, Akihito Endo and Carol A. Van Reenen 18.1 Introduction 239 18.2 Phylogeny 239 18.3 Morphology 240 18.4 Growth characteristics 240 18.5 Practical importance 241 18.6 Description of species 241

Section IV The family Lactobacillaceae 245 Giovanna E. Felis and Bruno Pot 19 The genus Lactobacillus 249 Bruno Pot, Giovanna E. Felis, Katrien De Bruyne, Effie Tsakalidou, Konstantinos Papadimitriou, Jørgen Leisner and Peter Vandamme 19.1 Historical background 249 19.2 Lactobacillus metabolism 250 19.3 The taxonomy of the genus Lactobacillus 282 19.4 The current phylogenetic structure of the genus Lactobacillus 286 19.5 Food and health applications of the genus Lactobacillus 293 19.6 Short descriptions of the validly published species of the genus Lactobacillus 294 19.7 Lactobacillus species awaiting validation pending publication of the manuscript (March 2013) 327 19.8 Lactobacillus species and subspecies that have been renamed after their original description 329 19.9 Lactobacillus species that have never been validly named, but whose names nonetheless appear in the literature, and their current names 335

20 The genus Paralactobacillus 355 Jørgen J. Leisner and Bruno Pot 20.1 Introduction 355 20.2 Defining the genus as phenotype and genotype 355 20.3 Biodiversity within the genus and species based on phenotype 356 20.4 Importance of the genus and particular species 356 20.5 Description of species 357

21 The genus Pediococcus 359 Charles M.A.P. Franz, Akihito Endo, Hikmate Abriouel, Carol A. Van Reenen, Antonio Gálvez and Leon M.T. Dicks 21.1 Historical background and chronology of nomenclature 359 21.2 Phenotypic differentiation of the genus Pediococcus 360 CONTENTS ix

21.3 Genotypic delineation of the genus Pediococcus 360 21.4 Phylogenetic structure within the genus Pediococcus 361 21.5 Isolation and cultivation 362 21.6 Identification of Pediococcus spp 362 21.7 Importance of the genus and particular species 365 21.8 Species of the genus Pediococcus 366

Section V The family Leuconostocaceae 377 Akihito Endo, Leon M.T. Dicks, Johanna Björkroth and Wilhelm H. Holzapfel 22 The genus Fructobacillus 381 Akihito Endo and Leon M.T. Dicks 22.1 Introduction 381 22.2 Phylogenetic relationships 381 22.3 Morphology 383 22.4 Biochemical characteristics 383 22.5 Physiological characteristics 386 22.6 Habitat 386 22.7 Species in the genus Fructobacillus 386

23 The genus Leuconostoc 391 Johanna Björkroth, Leon M.T. Dicks, Akihito Endo and Wilhelm H. Holzapfel 23.1 Historical background, chronology of nomenclature and relationship to other LAB 391 23.2 Definition of the genus as phenotype 392 23.3 Biodiversity within the genus based on phenotype 393 23.4 Genomic studies and genotyping of Leuconostoc 393 23.5 Importance of the genus and particular Leuconostoc species 394 23.6 Description of species of the genus Leuconostoc 395

24 The genus Oenococcus 405 Akihito Endo and Leon M.T. Dicks 24.1 Introduction 405 24.2 Phylogeny and evolution 405 24.3 Morphology 406 24.4 Growth characteristics 407 24.5 Intraspecies diversity 409 24.6 Practical importance 410 24.7 Stress response 410 24.8 Description of species in the genus Oenococcus 412

25 The genus Weissella 417 Johanna Björkroth, Leon M.T. Dicks and Akihito Endo 25.1 Historical background, chronology of nomenclature and relationship to other LAB 417 25.2 Defining the genus as phenotype and genotype 417 25.3 Biodiversity within the genus and within particular species based on phenotype 419 25.4 Importance of the genus and particular species 419 25.5 Descriptions of species in the genus Weisella 421

26 The genus Lactococcus 429 Wonyong Kim 26.1 Introduction 429 26.2 Defining the genus as phenotype and genotype 429 26.3 Biodiversity within the genus based on phenotype 433 26.4 Biodiversity within species based on phenotype 434 26.5 Importance of the genus Lactococcus and species 436 26.6 Description of species of the genus Lactococcus 437 x CONTENTS

Section VI The family 445 Maret du Toit, Melanie Huch, Gyu-Sung Cho and Charles M.A.P. Franz 27 The genus Lactovum 447 Harold L. Drake 27.1 Introduction 447 27.2 Phylogeny and taxonomy of Lactovum 447 27.3 Morphology of Lactovum 448 27.4 Soil: the origin of Lactovum 449 27.5 Growth properties and substrate range of Lactovum 449 27.6 Physiology of Lactovum 451 27.7 Genus description 452 27.8 Conclusion 453

28 The genus Streptococcus 457 Maret du Toit, Melanie Huch, Gyu-Sung Cho and Charles M.A.P. Franz 28.1 Historical background and chronology of nomenclature 457 28.2 Phenotypic differentiation of the genus Streptococcus 458 28.3 Genotypic delineation of the genus Streptococcus 458 28.4 Phylogenetic structure within the genus Streptococcus 459 28.5 Isolation and cultivation 465 28.6 Identification of Streptococcus spp. 466 28.7 Importance of the genus and particular species 475 28.8 Species of the genus Streptococcus 476

Section VII Physiologically ‘related’ genera 507 Wilhelm H. Holzapfel and Brian J.B. Wood 29 The genera Bifidobacterium, Parascardovia and Scardovia 509 Paola Mattarelli and Bruno Biavati 29.1 Historical background 509 29.2 Taxonomy of the bifidobacteria 514 29.3 Ecology 521 29.4 Health benefits 522 29.5 Industrial applications 523 29.6 Other applications 523 29.7 Description of species 524 29.8 Bifidobacterium: concluding remarks 534 29.9 The genera Parascardovia and Scardovia 534

30 The genus Sporolactobacillus 543 Stephanie Doores 30.1 Introduction 543 30.2 Defining the genus as phenotype and genotype 544 30.3 Importance of the genus and particular species 547 30.4 Description of species of the genus Sporolactobacillus 548

31 The genera Bacillus, Geobacillus and Halobacillus 555 Hikmate Abriouel, Nabil Benomar, Melanie Huch, Charles M.A.P. Franz and Antonio Gálvez 31.1 Introduction 555 31.2 The genus Bacillus 556 31.3 Related genera in the family Bacillaceae 563 31.4 Food, health and environmental applications 564 31.5 Concluding remarks 565 CONTENTS xi

32 The genera Halolactibacillus and Paraliobacillus 571 Kazuhide Yamasato and Morio Ishikawa 32.1 Introduction 571 32.2 The genus Halolactibacillus 571 32.3 Paraliobacillus ryukyuensis 578

Appendix: Guidelines for characterizing LAB, bifidobacteria and related genera for taxonomic purposes 583 Paola Mattarelli, Bruno Biavati, Walter Hammes and Wilhelm H. Holzapfel A.1 Introduction 583 A.2 Phenotypic criteria 584 A.3 Genotypic criteria 588 A.4 Additional criteria 589 A.5 Concluding remarks 591

Index 593

List of contributors

Hikmate Abriouel Departamento de Ciencias de la Salud, Paraje Las Lagunillas, s/n Edificio B-3, 23071-Jaén, Spain. Nabil Benomar Universidad de Jaén, Departamento de Ciencias de la Salud, Campus Las Lagunillas, s/n, E-23071- Jaén, Spain. Bruno Biavati Department of Agricultural Sciences, University of Bologna, via Fanin 42, 40127 Bologna, Italy. Johanna Björkroth Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, FIN-00014 Helsinki University,Finland. Gyu-Sung Cho Max Rubner-Institut, Federal Research Institute for Nutrition and Food, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany. Katrien De Bruyne Applied Maths NV, Keistraat 120, B-9830 Sint-Martens-Latem, Belgium. Leon M.T. Dicks Department of Microbiology, University of Stellenbosch, ZA-7600 Stellenbosch, South Africa. Stephanie Doores Department of Food Science, Penn State University, 432 Food Science Building, University Park, 16802, USA. Harold L. Drake Department of Ecological Microbiology, University of Bayreuth, D-95440 Bayreuth, Germany. Maret du Toit Institute for Wine Biotechnology, Stellenbosch University, Private Bag X1, Matieland, ZA-7602 Matieland, South Africa. Akihito Endo Department of Microbiology, University of Stellenbosch, 7600 Stellenbosch, South Africa; Functional Foods Forum, University of Turku, 20014 Turku, Finland. Giovanna E. Felis Department of Biotechnology, University of Verona, Strada le Grazie 15, I- 37134 Verona, Italy. Charles M.A.P. Franz Max Rubner-Institut, Haid- und Neu-Strasse 9, D-76131 Karlsruhe, Germany. Antonio Gálvez Departamento de Ciencias de la Salud, Paraje Las Lagunillas, Edificio B-3, E-23071-Jaén, Spain. Giorgio Giraffa Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CRA-FLC), 26900 Lodi, Italy. Cho Gyu-Sung Max Rubner-Institut, Haid- und Neu-Strasse 9, D-76131 Karlsruhe, Germany. Walter Hammes Talstr. 60/1, D-70794 Filderstadt, Germany. Kikue Hirota Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517,Japan. Wilhelm H. Holzapfel School of Life Sciences, Handong Global University, Pohang, Gyeongbuk, 7891-798, South Korea; Insheimer Strasse 27,D-76865 Rohrbach, Germany. Richard B. Hoover Athens State University, 300 North Beaty Street, Athens, Alabama 35611, USA. Lesley Hoyles Department of Microbiology, University College Cork, Cork, Ireland. Melanie Huch Max Rubner-Institut, Haid- und Neu-Strasse 9, D-76131 Karlsruhe, Germany. Morio Ishikawa Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, 1-1, Sakuragaoka 1-chome, Setagaya-ku, Tokyo 156-8502, Japan. Annelies Justé Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBeT), Department of Microbial and Molecular Systems (M2S), KU Leuven Association, Thomas More Mechelen, Sint-Katelijne-Waver, Belgium; Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium; Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium. Wonyong Kim Department of Microbiology, Chung-Ang University, 156-756 Seoul, Republic of Korea. Paul A. Lawson Department of Microbiology and Plant Biology, and Graduate Program in Ecology and Evolutionary Biology, University of Oklahoma, Norman, Oklahoma 73019, USA. xiv LIST OF CONTRIBUTORS

Jørgen J. Leisner Department of Veterinary Disease Biology, Faculty of Health Sciences, University of Copenhagen, Grønnegårdsvej 15, DK-1870 Frederiksberg C, Denmark. Bart Lievens Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBeT), Department of Microbial and Molecular Systems (M2S), KU Leuven Association, Thomas More Mechelen, Sint-Katelijne-Waver, Belgium; Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium; Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium. Oksana Lukjancenko Center for Biological Sequence Analysis, Department of Systems Biology, The Technical University of Denmark, Building 208, DK-2800 Kgs. Lyngby, Denmark. Paola Mattarelli Department of Agricultural Sciences, University of Bologna, via Fanin 42, 40127 Bologna, Italy. Kenji Nakajima Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517,Japan. Konstantinos Papadimitriou Laboratory of Dairy Research, Department of Food Science and Technology, Agri- cultural University of Athens, Iera Odos 75, 118 55 Athens, Greece; Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimioupolis-Zographou, 157 84 Athens, Greece. Elena V. Pikuta Department of Mathematical, Computer and Natural Sciences, Waters Hall, N204, Athens State University, 300 North Beaty Street, Athens, Alabama 35611, USA. Bruno Pot Lactic Acid Bacteria and Mucosal Immunology, Center for Infection and Immunity Lille, Institut Pas- teur de Lille, Université Lille Nord de France, CNRS, UMR 8204; Institut National de la Santé et de la Recherche Médicale, U1019, 1, Rue du Professeur Calmette, F-59019 Lille, France. Hans Rediers Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBeT), Department of Microbial and Molecular Systems (M2S), KU Leuven Association, Thomas More Mechelen, Sint-Katelijne-Waver, Belgium; Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium; Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium. Ulrich Schillinger Institute for Microbiology and Biotechnology, Max Rubner-Institut (MRI), Haid- und Neu-Str. 9, D-76131 Karlsruhe, Germany. Pavel Švec Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Tvrdého 14, 602 00 Brno, Czech Republic. Effie Tsakalidou Laboratory of Dairy Research, Department of Food Science and Technology, Agricultural Univer- sity of Athens, Iera Odos 75, 118 55 Athens, Greece. Peter Vandamme Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium. Carol A. Van Reenen Department of Microbiology, University of Stellenbosch, ZA-7600 Stellenbosch, South Africa. Trudy M. Wassenaar Molecular Microbiology and Genomics Consultants, Tannenstrasse 7, D-55576 Zotzenheim, Germany. Kris A. Willems Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBeT), Department of Microbial and Molecular Systems (M2S), KU Leuven Association, Thomas More Mechelen, Sint-Katelijne-Waver, Belgium; Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium; Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium. Brian J.B. Wood Formerly Reader in Applied Microbiology, Strathclyde Institute for Pharmacy and Biomedical Sciences, Arbuthnott Building, University of Strathclyde, Cathedral Street, Glasgow, Scotland, G4 0RE, UK. Kazuhide Yamasato Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, 1-1, Sakuragaoka 1-chome, Setagaya-ku, Tokyo 156-8502, Japan. Isao Yumoto Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517,Japan. Acknowledgements

Many people have contributed to this book’s production. Clearly we owe a great debt to the individuals and teams who have produced the chapters and the introductions to the sections into which those chapters are grouped. We do rec- ognize that they are busy people with careers to develop, and, generally, with an overcrowded schedule continuously filled with commitments. Moreover, the prevailing ethos in universities and research centres does not value producing such reviews as those presented here in the same way that original research publications are measured for promo- tion and career development. We are thus most fortunate to have assembled an outstanding team of internationally recognized experts who share our vision for a benchmark comprising well-crafted overviews of current developments against which future development may be measured. Although such benchmarks are essential for orderly development in any scientific discipline, this may apply to the LAB and their taxonomy in a very special way. Consequently, weare most grateful to all authors for their dedication, commitment and patience with the demands that we have placed upon them. Our task has been greatly assisted by the Wiley Blackwell staff who have worked with us at various stages in the project’s development, but we tender special thanks to Mr Andrew Harrison, who so expertly guided us through the last stages toward the manuscript ready to progress to actual book publication. We also wish to acknowledge the con- tributions made by Mr Robert Hine, who had to read through what must have seemed a very arcane text and identify errors, anomalies, miscited references and so much more, and who did so with great patience and good humour.

Wilhelm H. Holzapfel Brian J.B. Wood List of abbreviations

AFLP Amplified fragment length polymorphism ARDRA Amplified ribosomal DNA restriction analysis ATCC American Type Culture Collection BCCM Belgian Coordinated Collections of Microorganisms (www.bccm.belspo.be) BHI Brain-heart infusion medium BLAST Basic Local Alignment Search Tool BOX-PCR BOX-A1R-based repetitive extragenic palindromic PCR BRC Biological Resource Centre CBD Convention on Biological Diversity CCM Czech Collection of Microorganisms (http://www.sci.muni.cz/ccm/) CCUG Culture Collection, University of Göteborg, Sweden (http://www.ccug.se) CDMT 2-Chloro-4,6-dimethoxy-1,3,5-triazine CIP Collection of Institut Pasteur, France (http://www.pasteur.fr/ip/easysite/pasteur/en) CRISPR Clustered regularly interspaced short palindromic repeats DGGE Denaturing gradient gel electrophoresis DNA Deoxyribonucleic acid DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) Eh Redox potential EBRCN European Biological Resource Centres Network (www.ebrcn.net) ECCO European Culture Collection Organization (www.eccosite.org) EFFCA European Food and Feed Cultures Association EFSA European Food Safety Authority EMbaRC European Consortium of Microbial Resources Centres (www.embarc.eu) ERIC-PCR Enterobacterial repetitive intergenic consensus-polymerase chain reaction F6PPK Fructose-6-phosphate phosphoketolase FAFLP Fluorescent amplified fragment length polymorphism FAME Fatty acid methyl esters FDA (US) Food and Drug Administration FDP Fructose-1,6-bisphosphate FEMS Federation of European Microbiology Societies FOSHU Foods for Specified Health Use (Japan) γD10 Gamma-D10 value (decimal reduction value indicating the inactivation rate ofmicroorganisms by gamma radiation) GC Gas chromatography GIT Gastrointestinal tract GM Genetically modified GRAS Generally recognized as safe GTG-PCR (Primer) GTG5-polymerase chain reaction (PCR using 5′GTGGTGGTGGTGGTG3′as primer, suitable for the grouping of LAB) GYP Glucose-yeast extract-peptone-beef extract HAMBI HAMBI Culture Collection, University of Helsinki, Finland (http://www.helsinki.fi/hambi) LIST OF ABBREVIATIONS xvii

HMP Human Microbiome Project HPr H Protein (in the sugar phosphotransferase metabolic route) ICFMH International Committee on Food Microbiology and Hygiene of the IUMS (http://www.icfmh.org) IS Insertion sequence (in a genome) ISR Intergenic spacer region ITS Intergenic transcribed spacer IUMS International Union of Microbiological Societies (http://www.iums.org) JCFF Japanese Federation for Culture Collections JCM Japan Collection of Microorganisms, RIKEN BioResource Center (http://www.jcm.riken.jp) KCTC Korean Collection for Type Cultures (http://kctc.kribb.re.kr/English/index.aspx) LDH Lactate dehydrogenase MALDI-TOF Matrix-assisted laser desorption ionization-time of flight MIRCENs Microbial Resource Centres MLLE Multi-locus enzyme electrophoresis MLST Multi-locus sequence typing MLVA Multiple locus variable number of tandem repeats analysis MRS de Man–Rogosa–Sharpe medium (for selectively isolating certain lactobacilli) M-S Mitis Salivarius (agar) MUMi Maximal unique matches NAS Nalidixic acid + sulfamethazine (medium) NBRC NBRC Culture Collection, Japan (http://www.nbrc.nite.go.jp/e) NCBI National Center for Biotechnology Information (Bethesda, Maryland) NCIMB National Collection of Industrial, Food and Marine Bacteria, Scotland (http://www.ncimb.com) NCLS National Committee for Clinical Laboratory Standards (USA); now Clinical and Laboratory Standards Institute NCTC Public Health England Culture Collections (http://www.hpacultures.org.uk) NIH National Institutes of Health NLM National Library of Medicine (NIH, Bethesda) NRIC NODAI Research Institute (Japan) Culture Collection Center (http://nodaiweb.university.jp/nric) ORF Open reading frame OTU Operational taxonomic unit PBP Penicillin-binding protein PCR Polymerase chain reaction PCR-RFLP Polymerase chain reaction-restriction fragment length polymorphism PCU Pharmaceutical Sciences Chulalongkorn University Culture Collection, Thailand PFGE Pulsed-field gel electrophoresis PLC Phospholipase C QPS Qualified Presumption of Safety (EU) (adopted 2005: EFSA-Q-2004-021) RAPD-PCR Random amplification of polymorphic DNA-polymerase chain reaction RCA Reinforced clostridial agar REA-PFGE Restriction enzyme analysis with pulsed-field gel electrophoresis REP-PCR Repetitive element palindromic-polymerase chain reaction RNA Ribonucleic acid rRNA Ribosomal RNA SLSA Single-locus sequence analysis SNP Single nucleotide polymorphism SSCP Single-strand conformation polymorphism Sugar PTS Sugar phosphotransferase TGGE Temperature gradient gel electrophoresis TISTR Thailand Institute of Scientific and Technological Research (TISTR) Culture Collection Center (http://www.tistr.or.th/tistr_culture) TLC Thin layer chromatography tmRNA Transfer-messenger RNA TPY Trypticase Phytone Yeast tRNA Transfer RNA TSB Tryptic Soy Broth xviii LIST OF ABBREVIATIONS

TYC Trypticase-Yeast Extract-Cystine UKFCC United Federation for Culture Collections UKNCC United Kingdom National Culture Collection (www.ukncc.co.uk) USFCC United States Federation for Culture Collections WDCM World Data Center for Microorganisms (www.wdcm.org) WFCC World Federation for Culture Collections (www.wfcc.info) Abbreviations for genera and note on pronunciations

Standard abbreviation Genus

A Ab. Abiotrophia Ac. Atopococcus Ae. Aerococcus Af. Allofustis Ai. Alloiococcus Alk. Alkalibacterium Ap. Atopobacter At. Atopostipes B B.*Bacillus Bav. Bavariicoccus Bif.*Bifidobacterium C C. Carnobacterium Cat. Catellicoccus D D. Desemzia Dc. Dolosicoccus Dg. Dolosigranulum E E.*Escherichia Ent. Enterococcus Ere. Eremococcus F F. Facklamia Fru. Fructobacillus G G.*Geobacillus Glo. Globicatella Gra. Granulicatella xx ABBREVIATIONS FOR GENERA AND NOTE ON PRONUNCIATIONS

Standard abbreviation Genus

H H.*Halolactibacillus Hb. Halobacillus I Ig. Ignavigranum Is. Isobaculum L Lb. Lactobacillus Lc. Lactococcus Leuc. Leuconostoc Lg. Lacticigenium Lv. Lactovum M M. Marinilactibacillus Me. Melissococcus O O. Oenococcus P P.*Parascardovia Pa.*Paraliobacillus Ped. Pediococcus Pi. Pilibacter Pl. Paralactobacillus S S.*Scardovia Sp.*Sporolactobacillus Staph.*Staphylococcus Strep. Streptococcus T Tet. Tetragenococcus Tr. Trichococcus V V. Vagococcus W W. Weissella ∗Genus (phylogenetically) not a member of the LAB.

Note on pronunciations The etymologies of generic and specific names are in many cases supplied with a basic pronunciation of thenameas used by native speakers of standard English. Syllables are separated by full points, and the primary stressed syllable is indicated by a stress mark (′) following the stressed syllable. 1 Introduction to the LAB

Wilhelm H. Holzapfel1∗ and Brian J.B. Wood2 1School of Life Sciences, Handong Global University, Pohang, Gyeongbuk, South Korea; Insheimer Strasse 27, D-76865 Rohrbach, Germany 2Strathclyde Institute for Pharmacy and Biomedical Sciences, Strathclyde University, Glasgow, Scotland

1.1 The scope Lactic acid bacteria (LAB) have been intimately associated with human culture and well-being throughout history. In our time, the industrialization of food biotransformations and the positive attributes of particular microbes to sensory, quality and safety features of fermented foods have become synonymous with the positive image of LAB. Yet, the economic impact and role of LAB, both beneficial and detrimental, is as diverse as the six families, 36 genera andthe increasing number of species (>200 by the end of 2011) within the order Lactobacillales may suggest. The LAB belong to the Gram-positive bacterial phylum with ‘low’ (≤55 mol %) G+C in the DNA. They are grouped in the third class (Class III, the )oftheFirmicutes,withtheClostridia (Class I) and the Mollicutes (Class II) as the other two members. Based on comparative sequence analysis of the 16S rRNA gene, the Firmicutes are distinguished from the other Gram-positive phylum, the , with high mol % G+C(≥55 mol %) in the DNA. The two Gram-positive phyla comprise the following:

Phylum VIII: Firmicutes (Ludwig et al. 2009, modified)

• Class I: ‘Bacilli’ • Order I: Bacillales with 12 families, e.g.: • Family I: Bacillaceae; Family VII: ‘Sporolactobacillaceae’ (with one genus Sporolactobacillus) • Order II: ‘Lactobacillales’ with 6 families • Class II: ‘Clostridia’ • Class III: ‘Erysipelotrichia’ • ‘Class’ Mollicutes (cell wall-less): the Mycoplasmas

Phylum Actinobacteria (Ludwig et al. 2007) comprising more than 39 families and 130 genera (Ventura et al., 2007); examples:

• Coryneform and propionic acid bacteria; Bifidobacterium; Mycobacterium; Rhodococcus; Gardnerella • Filamentous representatives: Streptomyces and other Actinomycetes.

It is clear that, by phylogenetic definition, Bifidobacterium belongs to the Actinobacteria and not to the true LAB. Still we have included this and ‘related’ genera (see Chapter 29) in this book for historical and practical reasons, one being their beneficial effects on and association with the gut, and another that bifidobacteria physiologically resemblethe true LAB to some degree. Similar considerations seemed to justify the inclusion of Bacillus (Chapter 31) and ‘related’ genera (Chapter 32), in addition to the genus Sporolactobacillus (Chapter 30), all of which have some physiological

∗ Corresponding author email: [email protected]

Lactic Acid Bacteria: Biodiversity and Taxonomy, First Edition. Edited by Wilhelm H. Holzapfel and Brian J.B. Wood. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd. 2 CH1 INTRODUCTION TO THE LAB

Female urogenital track

Human gut ecosystem

Human environment Food environment – Food Fermentations

Figure 1.1 ‘Domestication’ of LAB in the human environment. The close relationship between human culture and the direct food environ- ment of humans probably partly supplied the microbial population of food fermentations, and vice versa features similar or comparable to the LAB. Bacillus infernus (e.g.) is a strict anaerobe that grows fermentatively on glucose (Boone et al., 1995). Bacillus coagulans is a thermophilic producer of pure lactic acid (Payot et al., 1999), while ‘probiotic’ strains of this species are being marketed under the name ‘Lactobacillus sporogenes’ (De Vecchi & Drago, 2006). Most species of the genus Geobacillus are reported to form catalase (Nazina et al., 2001), yet some strains of Geobacillus stearothermophilus (formerly Bacillus stearothermophilus ) have been found to be catalase negative (Holzapfel, unpublished results). Figure 1.1. The LAB are grouped in Order II, the ‘Lactobacillales’ (Garrity & Holt, 2001; Ludwig et al., 2009) under Class I (Bacilli)oftheFirmicutes. With presently six families and 40 genera, the LAB may be considered as ‘a rapidly expand- ing’ group of bacteria, especially when considering the rate at which the publication of new Lactobacillus and Strep- tococcus species occurs, with more than 150 (see Chapter 19) and 70 (Chapter 28) species, respectively. This wide taxonomic delineation of the LAB indeed suggests a wide diversity within this group, as is indicated in the division of the six families:

• ‘Aerococcaceae’ (with 7 genera); • ‘Carnobacteriaceae’ (with 16 genera); • ‘Enterococcaceae’ (with 7 genera); • Lactobacillaceae (with 3 genera); • ‘Leuconostoccaceae’ (with 4 genera); • Streptococcaceae (with 3 genera).

Table 1.1 summarizes information on the presently recognized families and genera, and a few selected ‘classical’ phenotypic characteristics of these genera. A highly interesting indication of biodiversity is the interpeptide bridge of the cell wall peptidoglycan of the LAB. At least five different peptidoglycan types have been reported, both for the relatively ‘small’ genus Alkalibacterium (with presently eight species) and the genus Weissella (presently 14 species). By contrast, only two peptidoglycan types are known for the genus Enterococcus (representing 43 species), and four types for the genus Lactobacillus (presently >150 species). Consensus on a comprehensive definition of ‘biodiversity’ probably does not exist, as it has to be delineated according to the scope or range under consideration. In terms of their biological diversity, the LAB have to be considered on the basis of taxonomic (genus, species and even strain) diversity, genetic diversity and phenotypic diversity in relation to an ecosystem and adaptation to extreme conditions. Even in earlier geological history, their physiological diversity and adaptation to a wide range of sometimes extreme habitats clearly suggested that the LAB are by no means a homogeneous group. Present-day phylogenetic approaches are valuable but do not necessarily explain the adaptation of particular LAB to specific ecological niches, and even less so the activation of adaptive survival mechanisms including stress factors. Diversity of the LAB is reflected by their association with diverse habitats, including niches with extreme conditions ranging from relatively high temperatures (around 50∘C) to low temperatures (0–2∘C), and also with examples of growth at high salt concentrations (up to 25% NaCl), low pH (around 3.9) and physiological bile salt concentrations (see Table 1.2). In contrast to other Gram- positive bacteria such as Bacillus or Listeria (relying on a global stress-response regulator such as σB), the LAB respond to stress with several conserved stress proteins, including DnaK, GroEL and Clp, which are also involved in cross- protection against different stress conditions. Moreover, the type of stress will determine whether other, more specific regulators or mechanisms will be utilized for protection against harmful conditions (Franz & Holzapfel, 2011).

CH1 INTRODUCTION TO THE LAB 3

h DNA the

+ Cin G % Mol 39–48 36–46 (continued overleaf) -Asp, -Glu, D D -Asp 41–42 -Thr-Gly; -Glu; cell wall D L L -Glu 34.6–36.2 -Asp, -Glu -Asp; type in the D D D D Peptidoglycan -Asp Dpm 32–44 -Lys-direct; -Lys direct 35–40.5 -Lys direct 35–44.4 -Lys- -Lys- -Lys- Orn- Lys(Orn)- L L L L ND 45–49 m ND 46–46.6 Orn- Orn- Lys(Orn)- Orn- L-D L L

) C 45 at Growth ∘ +

/(

C 10 at Growth ∘ +−

/ −− +− riiehydrolysis Arginine −− +−− ++ − b / / / − ++ −

ND

− NaCl ) a

++ −+ + c + rwhi 6.5% in Growth / / +− + / − /(

+

− + rwha H9.6 pH at Growth −− c / +++++ +++++ − + ND (?)

DL ) ) )ND e ofi.o lactate of Config. + + + ); ( ( ( ND ND ND ND ND ND ND + L L L ( L

)ND )

2 CO rmglucose from + +

/( /( Motility −− −− +− / / / +− +− −− −− −− −− + + − Morphology rods (pleomorph), single, pairs, chains pleomorph chains tetrads chains Section/ this book chapter in Chapter 12 Spherical cocci to oval/olive-like Chapter 9 Cocci, single, pairs, groups, Chapter 14 Cocci/rods .) Chapter 11 Straight rods, single, pairs .) Chapter 13 Rods, single, pairs, short chains M .), ( .), .), .) Chapter 10 Rods, single, pairs, chains .) .) c .), C Alk Dg .), .), ( .) .), ( ( .) Chapter 6 Cocci, also coccobacilli to .) .), Ig Lg .), Gra .), Bav .) Chapter 7 Cocci, ovoid, single, pairs, ( ( ( Ere Dc ( Tr Ac Al Ap Ab Glo ( At Is ( .), ( ( ( ( .) Chapter 8 Ovoid cocci, pairs, clusters, Ac ( ( .), ( ( F ( D Af ( ( ( Genus (abbreviation)

Selected ‘classical’ characteristics as key phenotypic features, and present grouping of the LAB Abiotrophia ‘Minor’ genera: Dolosicoccus Carnobacterium Eremococcus Globicatella Ignavigranum Alkalibacterium Aerococcus Facklamia ‘Minor’ genera: Allofustis Alloiococcus Trichococcus Marinilactibacillus Atopobacter Atopococcus Dolosigranulum Granulicatella Isobaculum Desemzia Atopostipes Bavariicoccus Lacticigenium

ScinIII /Section Carnobacteriaceae ScinII /Section Aerococcaceae Table 1.1 Family/ Section

4 CH1 INTRODUCTION TO THE LAB

h DNA the

+ Cin G % Mol 29–38 38–43 32–55 32.5–44.9 43–44 38–44 37–47 2 , 2 2 -Ala L -Ser, or -Ala -Ser. , ,or L L L 2 a 2 , D-ASP; ND 2 2–3 -Ala -Ala- -Ala -Ser- -Ser- cell wall L L L L L -Asp, -Asp or -Asp 37–42 -Asp 34.0–44.5 -Asp, -Ala -Ala-Gly- type in the D D D D D L L Peptidoglycan Dpm -Lys- -Lys- -Lys-Gly- -Lys- -Lys- -Lys- L ND 46 Lys- L Lys- L ND 34–40 Lys-Ala Lys- m Lys- Lys- L L L Lys-Ala, Lys-Ala Orn- Lys-Ala-Ser, Lys-Ser-Ala Lys-

a a a a C 45 at Growth ∘ + + + + − − / / / / − − −

a a a C 10 at Growth ∘ − + − / / / ND ND − +

)NDND a a riiehydrolysis Arginine + −+ + − + / − −+− / / /(

−

− NaCl a a

−+ + + rwhi 6.5% in Growth / −−+ −− −+−− +−−− / / + −

a a rwha H9.6 pH at Growth ) + −− +++ / + ND ND ND ND − ) +

)

DL )ND )NDND ) )( ) + ofi.o lactate of Config. )/ − − + + + DL ( ( ( ( ( ND ND D(L) − L L L D D )/DL/L( ( DL/L( − D D(

a

2 CO rmglucose from + − + / −

a a Motility d + + −+ −+ −− −− −− −− −+ / / − − − Morphology small groups rods straight to irregular, single, pairs and chains tetrads chains pairs or chains pairs, tetrads Section/ this book chapter in Chapter 18 Coccoid to pleomorph/curved .) Chapter 16 Spherical to ovoid cocci, single, .) Chapter 20 Rods Pl .) Chapter 22 Rods, single, pairs .) Chapter 23 Ovoid cocci in pairs or chains .), .) Chapter 15 Ovoid cocci in pairs or chains; Tet ( .), .) Chapter 21 Spherical cocci, single, pairs, .) Chapter 19 Short (coccobacilli) to long rods, ( .), Fru Me Lb Ent .) Chapter 24 Ellipsoid to spherical cocci in ( Cat ( .) Chapter 17 Ovoid cocci, single, pairs, chains Leuc Ap ( ( Ped ( O .) ( V ( ( .) Chapter 25 Short rods or ovoid; pairs, short ( ( Pi W ( ( Genus (abbreviation)

(Continued) Enterococcus Lactobacillus Fructobacillus Pediococcus ‘Minor genera’: Atopobacter Catellicoccus Melissococcus Pilibacter Weissella Paralactobacillus Tetragenococcus Leuconostoc Oenococcus Vagococcus

eto V Section VI Section

ScinIV /Section Enterococcaceae / Lactobacillaceae / Leuconostocaceae Table 1.1 Family/ Section CH1 INTRODUCTION TO THE LAB 5 .); Sp ( 34–43 33–46 Sporolactobacillus .); (Ser), H ( , n 1–3 -Asp, D ND 37.6 Lys- Lys-Ala Lys-Ala-Gly-Ala, Lys-Ser-Ala or Lys-Thr-Ala Lys-Ala Lys-Ala(Ser), Lys-Thr-Gly, Lys-Thr-Ala, Lys-Ser-Gly − − Halolactibacillus a a .); − Hb ( a + −− / a Halobacillus − / .); G ( −+ −−− −− − +− ) ) Geobacillus + + ( ( ND L L .); E ( a ) + /( Escherichia −− −− −− .); Bif ( Bifidobacterium .); B ( Bacillus . C. viridans .) Chapter 28 Ovoid cocci in pairs or chains .) Chapter 26 Ovoid cocci in pairs or chains . Strep ( Lc .) Chapter 27 Ovoid, slightly elongated; pairs ( , which grows at up to a pH of 10 and 7% NaCl. Lv ( .). Staph ( Lactococcus Streptococcus Lactovum

Weissella beninensis T. patagoniensis tetccaeeScinVII Streptococcaceae/Section All species arginine hydrolase positive except Exception: Some species/strains. Other genera referred to in this book are abbreviated as follows: Exception: Staphylococcus a b c d e 6 CH1 INTRODUCTION TO THE LAB

Table 1.2 Examples of growth/tolerance and association of LAB with extreme conditions. Strain variations may occur within a species (Holzapfel, unpublished data; see also Franz & Holzapfel, 2011)

Factor Value Organism Substrate/habitat

Low pH pH 2.8 Lb. suebicus Fermenting apple/pear mash pH 3.2 Lb. acetotolerans Rice vinegar pH ∼ 3.0 Lb. acidophilus ‘group’ Stomach/upper duodenum High pH pH 9.6 Ent. faecium Bacillus fermentation of soya Bile salts and pancreatic Physiological concentrations C. divergens Meat juice Lb. acidophilus ‘group’ Small intestines Lb. reuteri/Lb. paracasei Salt (NaCl) 18–24% Tet. muriaticus Salted fermenting fish 26.4% (tolerance) C. viridans From vacuum-packed bologna Low temperatures 0–2∘C Leuc. gelidum Chill-stored vacuum-packed meats Some carnobacteria High temperatures 55∘C Lb. delbrueckii subsp. delbrueckii Emmental-type cheese Up to 50∘C Strep. thermophilus Italian-type hard cheeses Lb. helveticus Nitrite >150 ppm Several LAB Cured meat Sorbic acid >2 g/kg Several LAB Preserved juices, etc. Hops resistance a Ped. damnosus Beer Ped. claussenii Lb. brevis Ethanol 15% Lb. fructivorans Isolated from ketchup 13% O. oeni Wine > b Radiation resistance γD10 = 1.0 kGy Lb. sakei Radurized meat Heat resistance D65 = 20–30min W. viridescens Processed meats Ent. faecalis aIn relation to ‘bitter hop compounds’ at concentrations ranging around 55 ppm of iso-ά -acids. bHigher resistance during exponential growth than in stationary phase (Hastings et al., 1986). C., Carnobacterium; Ent., Enterococcus; Lb., Lactobacillus; Leuc. Leuconostoc; O., Oenococcus; Ped., Pediococcus; Strep., Streptococcus; Tet., Tetragenococcus; W., Weisella.

Representatives of the LAB may be found in diverse habitats and under conditions defined by extreme intrinsic and extrinsic factors. Examples of the association of LAB with extreme conditions under which either growth or tolerance have been observed are given in Table 1.2. Different mechanisms may be basic to survival or adaptation to diverse habitats. Survival traits may either be determined by constitutive (‘intrinsic’) features of a species or a strain, or may depend on stress responses. Examples of the former may be associated, for example, with the (Gram-positive) cell wall properties, and with a stronger ability to maintain homeostasis in an environment with adverse conditions of high osmotic pressure or low pH. The ability to survive or adapt to extreme conditions also depends on stress responses, including tolerance to low or high temperatures or to bile salts. Stress responses may also involve resistance to envi- ronmental stresses typical of an ecosystem, for example physiological concentrations of pancreatic juice in the small intestine, or high salt concentrations typical of Asian fermented fish products. The adaptation of Lactobacillus suebi- cus, Lb. acetotolerans and the Lb. acidophilus ‘group’ (comprising, e.g., Lb. acidophilus, Lb. gasseri, Lb. crispatus and Lb. johnsonii) to low pH values around 3.0 is differentiated by the habitats typical of these species. The Lb. acidophilus ‘group’ is typically associated with the small intestine and the female urogenital tract (Hammes & Hertel, 2009), and may be able to either mildly ferment milk or at least survive fermentation by well-adapted species such as Strepto- coccus thermophilus. Strains of Lb. suebicus, isolated from mashes stored for up to a year, were found to grow at pH 2.5 and in the presence of 14% ethanol (Kleynmans et al., 1989), while Lb. acetotolerans was reported to grow even in fermenting rice vinegar broth and to tolerate 4–5% acetic acid at pH 3.5 (Entani et al., 1986). Carnobacterium viridans, originally isolated from vacuum-packaged sliced Bologna sausage, is an alkalitolerant species surviving even in satu- rated brine solution (Holley et al., 2002). The tetragenoccci are characterized by their high salt tolerance; an extreme example is Tetragenococcus muriaticus, strains of which are able to grow in the presence of 1–25% NaCl (Satomi et al., 1997). Strains of Lb. sakei, isolated from radurized meat, have shown, contrary to the ‘normal’ behaviour of bacteria, higher radiation resistance during exponential growth than in the stationary phase (Hastings et al., 1986). Extreme cold tolerance and ability to grow even at 1–1.5∘C was reported for Leuconostoc gelidum (Shaw & Harding, 1989) and some carnobacteria (Jones, 2004) isolated from vacuum-packed cold-stored meat. This extraordinary diversity in habitats and capacities to tolerate, and even thrive in, extreme conditions is in marked contrast with earlier impressions CH1 INTRODUCTION TO THE LAB 7 of the LAB as highly fastidious organisms that were very restricted in their environmental tolerances and possessing very exacting nutritional requirements. A rapidly increasing number of LAB genomes have been sequenced, and the information, in general, is being made publicly available. Comparative functional genomic analyses have become strong tools in support of a deeper under- standing of the mechanisms behind biodiversity and adaptation of LAB to diverse habitats. The trend of extensive gene loss or ‘ongoing reduction in genome size’, called ‘reductive evolution’ (Van de Guchte et al., 2006), combined with key gene acquisitions via horizontal gene transfer, may explain the specialization of LAB to a variety of nutritionally rich environments (Makarova et al., 2006; Makarova & Koonin, 2007; Schroeter & Klaenhammer, 2009). Adaptation of LAB to food and intestinal ecosystems is explained by genomic analyses revealing species-to-species variation in the number of pseudogenes, and functional genes directing metabolic ability and nutrient uptake. Even with a general trend of genome reduction, it appears that certain niche-specific genes have been acquired with location on plasmids or adjacent to prophages (Schroeter & Klaenhammer, 2009). An interesting example is the in silico analysis by Lebeer et al. (2008) of genome sequences reflecting differences between the cheese isolate Lb. helveticus DPC4571 (genome: 2,080,931 bp, with 1618 genes and 217 ‘pseudogenes’), and the closely ‘related’ probiotic strain Lb. acidophilus NCFM (genome: 1,993,564 bp, with 1864 genes but no ‘pseudo- genes’) from infant faeces. [The term ‘pseudogenes’ has been suggested for dysfunctional ‘relatives’ of known genes that have lost their protein-coding ability; they are considered to be neutral sequences ‘shaped by random muta- tions and chance events’ (Vanin, 1985; Andersson & Andersson, 2001; Kuo & Ochman, 2010)]. The suggested ‘loss of genes’ of Lb. helveticus DPC4571 is considered important for adaptation to the gut environment, while half of the phosphoenolpyruvate-dependent sugar phosphotransferases (PEP-PTS), cell wall-anchoring proteins, and all the mucus-binding proteins predicted for Lb. acidophilus NCFM were absent or classified as being ‘pseudogenes’ in Lb. helveticus DPC4571. Genes considered pivotal in suggesting the niche of a strain are thought to be involved in sugar metabolism, the proteolytic system and restriction modification enzymes. Of the nine niche-specific genes identified, six were dairy-specific genes identified for Lb. helveticus DPC4571 and encoded components of the proteolytic system and restriction endonuclease genes. The three gut-specific genes of Lb. acidophilus NCFM encoded bile salt hydrolase and sugar metabolism enzymes (O’Sullivan et al., 2009). Understanding the genomic information responsible for various phenotypes and their persistence and survival in specific ecosystems and niches is an exciting and rapidly expanding field of research in our time. Specific information on LAB genomics, with specific focus on functional (including probiotic) LAB is presented in Chapter 5. When discussing biodiversity of a specific group of microorganisms such as the LAB, the importance of taxonomyas a basis of communication is obvious. In this context, it was envisaged that the title (‘Lactic Acid Bacteria – Biodiversity and Taxonomy’) would suggest the intricate complexity of the interplay between biodiversity and taxonomy of this exciting group of bacteria.

1.2 A little history Pioneering contributions are frequently overlooked or even forgotten in our ‘post-modern’ era. This applies in a special way to the LAB and their key position in early microbiological studies in the 19th century. The second part of the 19th century is characterized by the advent of microbiology as a science. It is fascinating to note that some of the earliest studies on bacteria were conducted on various types of LAB, most of which were either of socio-economic or medical importance during that time. Probably the first starter cultures to be applied for industrial purposes were introduced in 1890, in Denmark, Germany and the USA, for the production of cheese and sour milk. This initiative laid the foundation for the development of the diverse branches of industrial microbiology and modern biotechnology. The early interest in LAB as microorganisms was prompted by practical issues related to the food and fermentation industries. Louis Pasteur can be considered as the father of microbiology and immunology, but was a chemist. His studies on the molecular structures of tartaric acid laid the foundations of stereochemistry. These were followed in the summer of 1856 by investigations on a problem with improper fermentation, where he detected lactic acid instead of the by-product alcohol. Subjecting the mixture to high temperature (‘pasteurization’) and thereby killing the microor- ganisms, enabled him to achieve a predictable fermentation by introducing pure microbial cultures (http://www.famous -scientists.net/Louis-Pasteur.html). The history of LAB taxonomy also reflects key developments and understanding around food spoilage and food fermentations, as exemplified for the genus Leuconostoc. Cienkowski (1878) was probably the first to detect strains of the genus Leuconostoc as spoilage organisms in sugar factories, where they were shown to produce a characteristic slime from sucrose. Although these strains were named Leuconostoc by the French botanist Van Tieghem (1878), Orla-Jensen (1919) disregarded this and used the generic name ‘Betacoccus’ in his approach to 8 CH1 INTRODUCTION TO THE LAB separate the LAB genera by phenotypic means at that stage. Bacterium gracili was isolated from wine and described by Müller-Thurgau (1908), and can be considered as a non-slime-producing Leuconostoc. The priority of the earlier name Leuconostoc was supported by later studies of McCleskey et al. (1947) and Niven et al. (1949), who described the isola- tion of non-slime-producing sucrose-fermenting strains of Leuconostoc from sausages. During the late 1940s the biolog- ical and ecological diversity of representatives of the genus Leuconostoc became clear, and was underlined by further studies, including those by Pederson & Ward (1949), describing slime-producing strains from fermenting cucumbers. Lister (1878) is recognized for several pioneering contributions to the understanding of sepsis and antisepsis in health services and the application of phenol for treating wound infections. Less well known is his discovery, regarding LAB, that milk clotting is caused by ‘Streptococci’, and, moreover, the first isolation, in 1873, of a pure culture he called ‘Bacterium lactis’(Lactococcus lactis) (http://de.wikipedia.org/wiki/Joseph_Lister,_1._Baron_Lister). In 1973 a Symposium was held between the 19th and 23rd of September in beautiful autumn weather at the Long Ashton Research Station of the University of Bristol. It was organized by Drs J.G. Carr, C.V.Cutting and G.C. Whiting, and titled ‘Lactic Acid Bacteria in Beverages and Food’. The resulting book of the proceedings (Carr et al., 1975) claimed on its dust jacket that it was ‘the first comprehensive review of lactic acid bacteria to be published inasingle volume’. This was not strictly accurate as it focused on organisms associated with the industries delineated in the title, and thus there was little said about organisms such as most members of the genus Streptococcus. Despite this limitation, the meeting otherwise represented with reasonable accuracy the organisms comprising the Lactic Acid Bacteria (LAB), as they were known at that time . The same could be said of the Symposium participants (115 in number) as representatives of the scientific community active in studying the organisms at that time, and thelistof ‘Participants in the Symposium’ reads, at least in part, like a roll call of the pioneers in modern study of the group. There were 23 presentations divided into six Sections, plus opening and closing addresses. Preceding the 1973 Symposium was the milestone ‘Symposium on Lactic Acid Bacteria’ conducted during the 52nd annual meeting of the Society of American Bacteriologists at Boston, 28 April 1952, and convened by Ralph Tittsler (Tittsler et al., 1952). One of the valuable contributions of this symposium was to correct controversies in existing LAB nomenclature, confirming (e.g.) Betacoccus as Leuconostoc, and referring to early controversial opinions regard- ing ‘Lactobacillus bifidus’ (first suggested by Tissier, 1899) and its assignment to a new ‘non-butyric acid producing anaerobic genus’ (Orla-Jensen et al., 1936; Pederson, 1945; Tittsler et al., 1952). A special (and frequently underesti- mated) feature of the ‘early’ studies on LAB was the meticulous investigations and detailed reports on their physiology, with emphasis on growth factors, growth temperature ranges, and niche-specific physiological activities with regard to fermentation and spoilage. Of particular interest in the context of the volume published on the 1973 Symposium is the number of LAB genera and species referred to in the index to the book. There were six genera and in total 65 species. The genus Lactobacillus dominated with 45 species, but the ambiguous position of the genus Bifidobacterium was illustrated by there being only one reference to the genus (no species mentioned) but four references to ‘Lactobacillus bifidus’. The only reference in the index to ‘Genetic Code’ leads to a 4-page section on ‘Mean Base Composition and Homology of DNA’. This conference surely represents the benchmark against which we can measure the growth in our understanding of the LAB in the intervening 36 years. Exactly 10 years later the first of the now triennial LAB conferences organized by the Netherlands Society forMicro- biology and originally centred at Wageningen University in The Netherlands (which is still pivotal to the continuing success of these important meetings) took place in the University. It was called ‘Lactic Acid Bacteria in Foods; Genet- ics, Metabolism and Applications’, with each of the topics in the subtitle being assigned a day, although, because of other activities, the main sessions actually had a morning each to themselves. There were over 200 people present, with 68 contributions drawn from 18 countries. The organizers chose deliberately to omit Classification as a topic for the meeting. Unfortunately there is no index to the special 1983 edition of the Antonie van Leeuwenhoek Journal of Microbiology (49: 209–352) containing the plenary papers, while the short presentations were presented as photo- copies in a ring binder; fast, efficient, but we cannot present the sort of statistics given for the 1973 meeting concerning numbers of validly recognized genera and species. The most striking thing about the whole conference is probably the extent to which genetics had started to move centre-stage, with plenary papers on genetic transfer systems in LAB, functional properties of their plasmids and ‘The bacteriophages of LAB with emphasis on genetic aspects of group N lactic streptococci’. It would be tedious and probably pointless to present a discussion of each of the intervening LAB Conferences (the latest one, in 2011, was the 10th in the Dutch series) but anyone who has attended the more recent meetings will readily appreciate how the field has grown and changed. We may also note that for several years there were LAB conferences held in France, in the historic town of Caen, and organized under the auspices of Adria Normandie. These tended to focus more on applications of the bacteria, and were a nice counterpoint to the Netherlands congresses. The other great change of consequence for the present work is the massive increase in the number of both genera and